All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.
G protein-coupled receptors are prevalent throughout the human body, comprising approximately 60% of known cellular receptor types. They mediate signal transduction across the cell membrane for a very wide range of endogenous ligands and consequently participate in a diverse array of physiological and pathophysiological processes, including, but not limited to, those associated with cardiovascular, central and peripheral nervous system reproductive, metabolic, digestive, immunoinflammatory, and growth disorders, as well as other cell regulatory and proliferative disorders. Agents which selectively modulate functions of G protein-coupled receptcs have the potential for therapeutic applications. These receptors are becoming increasingly recognised as important drug targets, due to their crucial roles in signal transduction (G protein-coupled receptors, IBC Biomedical Library Series, 1996)
One of the most intensively studied G protein-coupled receptors is the receptor for C5a. C5a is one of the most potent chemotactic agents known, recruiting neutrophils and macrophages to sites of injury, altering their morphology; inducing degranulation; increasing calcium mobilisation, vascular permeability (oedema) and neutrophil adhesiveness; contracting smooth muscle; stimulating the release of inflammatory mediators, including histamine, TNF-α, IL-1, IL-6, IL-8, prostaglandins, and leukotrienes, and of lysosomal enzymes; promoting the formation of oxygen radicals; and enhancing antibody production (Gerard and Gerard, 1994).
Overexpression or underregulation of C5a is implicated in the pathogenesis of immune system-mediated inflammatory conditions, such as rheumatoid arthritis, adult respiratory distress syndrome (ARDS), systemic lupus erythematosus, tissue graft rejection, ischaemic heart disease, reperfusion injury, septic shock, psoriasis, gingivitis, atherosclerosis, Alzheimer's disease, lung injury and extracorporeal post-dialysis syndrome, and in a variety of other conditions (Whaley 1987; Sim 1993).
Agents which limit the pro-inflammatory actions of C5a have potential for inhibiting chronic inflammation, and its accompanying pain and tissue damage. For these reasons, molecules which prevent C5a from binding to its receptors are useful for treating chronic inflammatory disorders driven by complement activation. Such compounds also provide valuable new insights into the mechanisms of complement-mediated immunity.
In our previous applications No. PCT/AU98/00490 and Australian provisional No. PR8334, the entire disclosures of which are incorporated herein by this reference, we described the three-dimensional structure of some analogues of the C-terminus of human C5a, and used this information to design novel compounds which bind to the human C5a receptor (C5aR), behaving as either agonists or antagonists of C5a. It had previously been thought that a putative antagonist might require both a C-terminal arginine and a C-terminal carboxylate for receptor binding and antagonist activity (Konteatis et al, 1994). In PCT/AU98/00490 we showed that in fact a terminal carboxylate group is not generally required either for high affinity binding to C5aR or for antagonist activity. Instead we found that a hitherto unrecognised structural feature, a turn conformation, was the key recognition feature for high affinity binding to the human C5a receptor on neutrophils. As described in our Australian provisional application No. PR8334, filed on 17th Oct. 2001, we used these findings to design constrained structural templates which enable hydrophobic groups to be assembled into a hydrophobic array for interaction with a C5a receptor. We have subsequently found that preferred compounds of this class are able to inhibit inflammatory bowel disease, osteoarthritis, and hypersensitivity states, and this is described in our Australian provisional applications No. 2002952084, filed on 16th Oct. 2002, No. 2002952086, filed on 16th Oct. 2002, and No. 2002952129, filed on 17th Oct. 2002 respectively. The entire disclosures of these specifications are incorporated herein by this reference.
Fibrosis, the ingrowth of fibroblasts and the production of extracellular matrix to form abnormal scarring, can result from many causes, including trauma, surgical interventions, infections and pathological conditions. Fibrosis is a sequel of conditions such as chronic inflammation, including inflammation arising from diabetes and hypertension, but can arise in the absence of inflammation. It can occur in a variety of tissues, including but not limited to the lung, kidney, liver and heart. Fibrosis contributes to the loss of function experienced in such conditions, through the formation of abnormal quantities of extracellular matrix which change the physical properties of the scarred tissue. Diabetes- or hypertension-induced fibrosis of the heart, for instance, produces stiffening of the ventricle walls which contributes to decreased cardiac output.
It is estimated that 45% percent of deaths in the USA are attributable to disorders exhibiting proliferative fibrosis. Although fibrosis is debilitating and may be life-threatening, and the number of individuals who may benefit from an effective antifibrotic therapy is large, currently there are no effective treatments available. Both acute and chronic diseases which induce inflammation in the lung can lead to an irreversible process characterized by pulmonary fibrosis (PF). Pulmonary fibrosis may also occur as a side-effect of treatment with chemotherapeutic agents such as bleomycin. Pulmonary fibrosis is a severe disease, which leads to functional impairment and death. Cardiac fibrosis is associated with chronic hypertension, and both cardiac and renal fibrosis are long-term sequelae of diabetes.
Fibrosis is a dynamic process, and is considered to be potentially reversible. The extracellular matrix laid down during fibrosis may be resorbed after the withdrawal of the fibrotic stimuli. In many cases, however, the presence of fibrosis is only identified after loss of function has already taken place, for instance where decreased cardiac output is a sign of otherwise undetected cardiac fibrosis. Consequently, while it is desirable in certain circumstances to be able to prevent fibrosis from occurring, it is also desirable to be able to reverse existing fibrosis once it is detected. However, current therapeutic options for the treatment of fibrotic conditions are limited and relatively ineffective (el-Nahas et al. 1997).
The effects of drug-induced ad hypertension-induced pulmonary and renal fibrosis in animal models can be prevented or partially reversed by compounds which act by suppressing inflammatory events and down-regulating lung pro-collagen I over-expression (Iyer et al., 1999a,b).
We have shown that the administration of pirfenidone or spironolactone can prevent and partially reverse cardiac fibrosis and the increase in cardiac stiffness which occurs in streptozotocin-induced diabetes in rats (Miric G, et al., 2001) It is thought that pirfenidone acts by inhibiting increased TGF-β mRNA expression, allowing an increase in expression of metalloproteases which degrade the collagen I laid down during fibrosis. The mode of action of spironolactone is at present unknown. Spironolactone is a steroid analogue which is primarily used as a diuretic; pirfenidone (5-methyl-1-phenyl-2-(1H)-pyridone), an investigational compound being investigated as an anti-fibrotic agent in a number of indications.
It would be highly desirable to identify other therapeutically or prophylactically active agents for use in the treatment or prevention of fibrosis.
The overexpression or underregulation of a G-protein-coupled receptor, the C5a receptor, has been implicated in immune-system mediated events such as inflammation. Agents which influence C5a receptor activity, such as C5a receptor antagonists, have the potential to mediate inflammatory events, and may provide a means of therapeutic or prophylactic intervention, but have not previously been suggested as potential agents in the treatment or prevention of fibrosis.
We have now surprisingly found that a cyclic peptide with C5a receptor antagonist has the ability to ameliorate cardiac fibrosis in an animal model of this condition.